On the Chemical Constitution of the 

Proteins of Wheat Flour and Its 

Relation to Baking Strength 



A THESIS SUBMITTED TO THE FACULTY OF THE GRADUATE 
SCHOOL OF THE UNIVERSITY OF MINNESOTA 

BY 

MORRIS J. BLISH 

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR 
THE DEGREE OF DOCTOR OF PHILOSOPHY 

June, 19 15 



Easton, Pa.: 

EscHENBACii Printing Co. 

1916 



On the Chemical Constitution of the 

Proteins of Wheat Flour and Its 

Relation to Baking Strength 



A THESIS SUBMITTED TO THE FACULTY OF THE GRADUATE 
SCHOOL OF THE UNIVERSITY OF MINNESOTA 

BY 

MORRIS J. BUSH 

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR 
THE DEGREE OF DOCTOR OF PHILOSOPHY 

June, 1915 



Easton, Pa.: 

EscHENBACH Printing Co. 

1916 






^in 



ON THE CHEMICAL CONSTITUTION OF THE PROTEINS 

OF WHEAT FLOUR AND ITS RELATION TO 

BAKING STRENGTH 

By M. J. Bush 

Received June 7, 1915 

INTRODUCTION 

The most generally accepted, definition of "baking 
strength" of a wheat flour is that put forward by- 
Humphries and BifTen/ in 1907, which states that a 
"strong wheat is one which yields flour capable of 
making large, well-piled loaves;" a definition similar 

to that of Jago," who states that "strength is 

defined as the measure of the capacity of the flour for 
producing a bold, large-volumed, well-risen loaf." 
Since the value of wheat (other things being equal) 
depends on the so-called "strength" of the flour which 
may be made from it, it is obviously of great importance 
that complete knowledge be obtained concerning the 
factors which cause strength, and to this end an 
enormous amount of scientific, work has been done, 
especially during the last twenty years. In spite of 
the fact that some of the foremost investigators of 
the world have bent their energies to this task, the 
problem is not yet completely solved, although con- 
siderable light has been thrown on the subject. It is 
not yet possible to correlate baking strength with any 
chemical or physical factor to such an extent that a 
simple laboratory test or group of tests will always 
furnish an infallible guide, but it is necessary to mill 
the wheat into flour and have a sample of it actually 
baked into a loaf of bread by an expert baker, before 
its strength can be accurately ascertained. 

FACTORS WHICH MAY INFLUENCE BAKING STRENGTH 

Almost every known constituent, or group of con- 
stituents, and almost every known physical and chemical 
property of flour has been investigated with respect 
to its possible relation to baking strength, but as yet 
no one is believed to have discovered a limiting factor 
or group of factors which completely solves the prob- 
lem. Moreover, there is a general disagreement 
among many of the leading investigators as to the 
importance which should be attached to each factor 

1 "The Improvement of English Wheat," Jour. Agr. Set., 2 (1907), 
1-16. 

2 "Technology of Bread Making." Chap. XV, p. 291, 1911. 



4 
or set of factors, and two workers frequently have 
arrived at exactly opposite conclusions after having 
investigated practically the same problem; however, 
much of this confusion is caused by the use of different 
methods of analysis. 

A brief review of some of the more important work 
which has been done will serve to bear out the pre- 
ceding statement, as well as to indicate the many sides 
from which the question of flour strength has been 
studied. 

GLIADIN-GLUTENIN RATIO — As soon as Osbornc and 
Voorhees,^ in 1893, established the composition and 
properties of the wheat proteins, attention was at- 
tracted to gliadin and glutenin, the two conspicuous 
and characteristic proteins of wheat, which were 
shown to make up the gluten, the more or less elastic 
binding material which enables flour to be made into 
the dough with its characteristic elastic, gas-retaining 
property, and which may be separated from the starch 
and soluble proteins by the well-known process of 
washing the dough in a stream of water. Fleurent,' 
in 1896, claimed that flour strength depends on the 
proportion of gliadin to glutenin present in the gluten 
of the flour. He concluded from his experiments 
that the optimum ratio was 75 parts of gliadin to 25 
of glutenin or 3:1. He assigned certain limits, outside 
of which flours were said to be of poor baking quality. 
Snyder,^ in 1899, published similar results, although 
he fixed his ideal ratio at 65:35. He also states* 
that the quality rather than the quantity of gluten is 
the important factor, because he was able to add up 
to 20 per cent starch to flour without decreasing its 
baking quality. Regarding the quantity of gluten in 
flour, the amount of gliadin present, the ratio of gliadin 
to glutenin, and the relation of these to baking quality, 
it suffices to say that the results of different investi- 
gators quite frequently are not concordant. However, 
as mentioned before, this is in a considerable measure 
due to different analytical methods employed by differ- 
ent workers. 

CRUDE GLUTEN — The crude gluten determination, 
which consists essentially of washing the gluten free 
from starch and soluble material by means of water, 
and weighing the gluten, both in a wet and dry state, 

1 "The Proteids of the Wheat Kernel," Am. Chem. J., 15 (1893), 
392-471; Ibid., 16 (1894), 524-535. 

* "Sur une method chimique d'appreciation de la valeur boulangere 
des farines de ble," Compi. rend.. 123 (1896), 755-758. 

» Minn. Exp. Sla. Bull., 62 (1899). 

« U. S. Dept. Agr., Bull. 101 (1901). 



5 
was for a long time considered of great value, but 
Snyder and Norton,^ in 1906, Chamberlain, ^ in 1906, 
and others showed that it gave but little information 
which might not be gained from a determination of 
total nitrogen or alcohol-soluble nitrogen. Neverthe- 
less, it is still used extensively by millers and bakers, 
and in technical laboratories. 

PHYSICAL STATE OF GLUTEN AND SUGAR CONTENT 

In 1907, Wood^ published the results of a thorough 
and systematic study of the chemistry of flour strength. 
He concluded that there is no difference in the chemical 
constitution of gliadin and glutenin from strong and 
weak flours, and decided that strength (particularly 
shape of loaf) is much more closely related to the 
physical state of gluten, which in turn is profoundly 
affected by the presence of electrolytes. He showed 
that minute quantities of acids and bases tend to 
"disperse" gluten, making it weak and inelastic, 
while small quantities of neutral salts have the opposite 
and consequently beneficial effect. Furthermore, he 
found that the volume of a loaf of bread is proportional 
to the rate of carbon dioxide evolution resulting from 
diastatic activity of yeast in the later stages of fer- 
mentation. In other words, he concludes that loaf 
volume depends on the amount of available sugar in 
the later stages of fermentation. Alway and Hartzell,.^ 
in 1909, however, performed experiments which led 
them to say, in contrast to Wood's findings, "there is 
clearly no direct connection shown between the size 
of the loaf and the volume of gas evolved. The thir- 
teen fiours which gave the largest loaves evolved on 
the average somewhat less gas than the other thirteen 
flours." Shutt^ states that from his experimental 
evidence he was unable to find any relation between 
size of loaf and sugar content. 

ENZYMES — Comparatively less study has been made 
of the enzymes of flour and their relation to strength. 
Perhaps the most prominent work in this field is that 
which was done simultaneously but independently by 
Baker and Hulton,^ and by Ford and Guthrie,^ in 

1 "Crude Gluten," J. Am^ Chem. Soc, 28 (1906), 8-25. 

2 "Properties of Wheat Proteins," Ihid., 28 (1906), 1657-1667. 

3 "The Chemistry of Strength of Wheat Flour," Jour. Agr. Sci., 2 
(1907), 139-161 and 267-277. 

< Neb. Exp. Sta.. 23rd Annual Report, 1909. 

' "Flour — the Relationship of Composition to Bread Making Value," 
Canadian Miller and Cerealist, 5 (1913), 176-178. 

' "Conditions Affecting the Strength of Wheaten Flour," Jour. Soc. 
Chem. Ind.. 27 (1908), 368-376. 

' "The Amylolytic and Proteolytic Ferments of Wheaten Flour and 
Relation to Baking Value." Jour. Soc. Chem. Ind., 27 (1908), 389-393. 



igoS. They point out that both proteoclastic and 
amyloclastic enzymes are present in flour and in many 
instances may exert a profound influence on its bread- 
making qualities. Baker and Hulton state that 
"it is obvious that the strength of a flour must be 
closely connected with the gluten, although no doubt 
the presence of enzymes, soluble carbohydrates, and 
mineral constituents all play a part." Koch,^ in 
1914, found no difference in the quantity of diastase 
in strong and weak flours, after extracting them with 
water at 0° according to the method of Thatcher 
and Koch. 2 

CONCENTRATION OF HYDROGEN IONS H. JcSSCn- 

Hansen,' in 1911, finds a close relationship between 
the concentration in hydrogen ions and baking strength, 
and asserts that there is an optimum hydrogen-ion 
concentration for flour, the poorer flours having lower 
concentrations. He. attributes the beneficial effects 
of neutral salts and "flour improvers" on flour to the 
fact that they raise the hydrogen ion concentra- 
tion. 

SOLUBLE PROTEINS — There does not seem to have 
been a very* considerable amount of work done regard- 
ing the role of the soluble proteins as a factor in baking 
strength. Snyder,^ in 1897, says "When any of the 
wheat proteids except gliadin or glutenin are extracted 
the expanding and bread-making qualities of the flour 
are not affected." The conclusions of Bremer,^ 
in 1907, are also to the effect that the soluble proteins 
have little bearing on flour strength. Rousseaux 
and Sirot,^ in 1913, consider the ratio of total nitrogen 
to soluble nitrogen as a valuable index to baking value 
and have determined an ideal ratio for flours according 
to their method, as well as the limits between which 
strong flours must fall in this respect. 

GENERAL CONSIDERATIONS— Numerous othcr results 
of careful and valuable research might be cited, but 
the above serve to indicate the confusion existing in 

' "The Diastase and Invertase Content of Wheat Flour and Their 
Relation to Baking Strength." Thesis for Master's Degree, University of 
Minnesota, June, 1914. 

2 "The Quantitative Extraction of Diastases from Plant Tissues," 
J. Am. Chem. Soc. 36 (1914), 759-770. 

' "Studies on Wheat Flour. Influence of H-ion Concentration on 
Baking Value of Flour," Compl. rend.. 10 (1911), 170-206. 

* Minn. Exp. Sla. Bull.. 54 (1897). 

' "Hat der gehalt des Weizemehles an Wasserloslichen StickstoEf einer 
Einfluss auf seiner Backwert," Ztschr. Unter. Nahr. Genuss., 13 (1907), 
69-74 

5 "Les matiSres azotees solubles comme facteur d'appreciation des 
farines," Compt. rend. Acad. Sci., 156 (1913), 723-725. 



7 



the present state of our knowledge regarding the 
factors involved in flour strength, and is intended to 
serve this purpose rather than constitute anything 
like a complete summary of all the work which has 
been done in this field. Numerous summaries of this 
sort have been published in text-books and articles 
dealing with methods of milling and baking technology, 
such as that of the Jagos,i and a repetition of them 
here would serve no useful purpose. It is believed, 
moreover, that the above discussion indicates nearly 
all of the view-points from which the problem of the 
chemistry of flour strength has been attacked. The 
situation is very well expressed by Bailey^ when he 
says: "Perhaps one of the reasons that a greater de- 
gree of success has not attended these endeavors is 
the fact that it has been attempted to discover one 
constituent (or group of constituents) which is the sole 
determining factor. It does not seem reasonable to 
believe that in so complex a substance as wheat flour 
the percentage of one constituent can be regarded as 
solely indicative of baking quality. Rather must we 
study these various compounds in their relation to 
one another, in an effort to arrive at their single and 
combined effects." 

PURPOSE OF THIS INVESTIGATION 

In a series of investigations of the various factors 
which may influence the strength of wheat flour, now 
in progress in the Division of Agricultural Chemistry 
of the University of Minnesota, it was proposed to 
study the chemical constitution of the various proteins 
in flour with a view toward ascertaining more defi- 
nitely than has yet been done, whether or not the pro- 
teins of a strong flour may differ in their chemical 
constitution from those of a weak flour, since the 
physical properties of their glutens are found to differ 
so markedly. 

Wood, 3 in 1907, following Osborne and Harris' 
modification of Hausmann's method, subjected samples 
of gliadin and crude gluten (composed chiefly of gliadin 
and glutenin) of flours of different strength, to hydrolysis 
for 8 hours with strong hydrochloric acid. He then 
steam-distilled the products of hydrolysis with magnesia 
and determined the percentage of nitrogen given off 
as ammonia. Finding a close agreement in the different 
samples he concluded that gliadin and glutenin of 

1 Loc. cit. 

2 "Relation of the Composition of Flour to Baking Quality," Canadian 
Miller and CerealisI, 5 (1913), 208-209. 

9 Loc. cit. 



all wheat flours are of the same chemical composition, 
since the work of Wood/ a more detailed method of 
protein analysis, which gives further insight into the 
constitution of the protein molecule and is capable of 
yielding quantitative results, has been presented by 
Van Slyke,2 who has incidentally shown that the 
hydrolysis of gliadin with strong hydrochloric acid 
is not complete at the end of 8 hours. It was therefore 
decided to make further study of the chemical con- 
stitution of flour proteins in the light of better methods 
of analysis now available. 

METHODS OF STUDYING CHEMICAL COMPOSITION OF 
PROTEINS 

It has been shown repeatedly that for practical 
considerations all of the nitrogen of flours of the higher 
milling grades may be regarded as in the proteins. 
The chemical structure of the proteins has been clearly 
demonstrated by Fischer^ and a host of other workers 
since, so that it needs no elaborate discussion here. 
Briefly stated, the facts appear to be that the protein 
molecule is made up of a number of amino acids, there 
being some i8 or 20 of these which occur in natural 
proteins. These are probably linked together by 
anhydride combinations between the amino group of 
one amino acid and the carboxyl group of another. 
This is indicated by the nature of the products formed 
(amino acids) when the protein is subjected to hydroly- 
sis. Moreover, it appears that the characteristic 
chemical and physical nature of individual proteins 
depends largely on the nature and number of the 
various amino acids of which they are composed. 
In a comparison of the chemical constitution of proteins, 
then, it is necessary to split the molecule by hydrolysis 
into its "bausteine" (characteristic units) and determine 
the relative proportions of these which are formed in 
each case. There is no known method of ascertaining 
the exact manner in which these units are grouped 
together in the various proteins, since even the sensi- 
tive anaphylaxis reaction is not specific in the case of 
many vegetable proteins, as has been demonstrated 
by Wells and Osborne,* who found that animals 

1 Loc. cit. 

2 "The Analysis of Proteins by the Determination of the Chemical 
Groups Characteristic of the Different Amino Acids," J. Biol. Chem.. 10 
(1911). 15-55. 

' "Untersuchungen uber Aminosauren, Polypeptide und Proteine," 
Berlin, 1899-1906 

4 "Is the Specificity of the Anaphylaxis Reaction Dependent on the 
Chemical Constitution of the Proteins or on Their Biological Relations? 
The Biological Reactions of the Vegetable Proteins. II," Jour. Infect. 
Dis.. 12 (1913), 341-358. 



9 
sensitized with gliadin of either wheat or rye will react 
with hordein of barley, a protein known to have a 
different chemical constitution, and that gliadin and 
glutenin, known to be different as regards the relative 
proportions of the various amino acids in their molecule, 
react anaphylactically with each other. 

METHOD USED IN DETERMINING PRODUCTS OF PROTEIN 
HYDROLYSIS 

Van Slyke's method gives the most detailed insight 
into the protein molecule of any known method which, 
at the same time, indicates quantitatively the dis- 
tribution of its component units. Accordingly, the 
Van Slyke method, in some cases slightly modified, 
was used in this investigation. The method, which 
is an extension of the principle of the Hausmann 
method, consists of a division of the protein molecule 
into various groups, or units, after prolonged hy- 
drolysis with hydrochloric acid, and the determination 
of the percentage of nitrogen in each individual group, 
thus ascertaining the distribution of the total nitrogen 
in the protein. Briefly, the groups determined are: 
(i) ammonia or amide nitrogen, which is considered 
to be derived from — CONH2 or — CONHOC — 
groups linked to the carboxyl groups of the dicarboxylic 
acids in the protein molecule (glutamic and aspartic 
acids); (2) humin nitrogen, from the dark-colored 
pigment and slight amount of insoluble matter always 
formed in the hydrolytic products of acid hydrolysis 
of proteins; (3) the amino nitrogen of the mono-amino 
acids, which corresponds to all of the mono-amino 
acids excepting proline and oxy-proline; (4) the non- 
amino nitrogen of the mono-amino acids, which corre- 
sponds to the proline and oxy-proline; and (5) to (8) 
the nitrogen corresponding to each of the individual 
di-amino acids, i. e., arginine, lysine, histidine, and 
cystine, respectively. Thus, eight units of the protein 
molecule may be estimated quantitatively, the de- 
termination of histidine nitrogen and lysine nitrogen 
being subject to a larger experimental error than the 
other units, which may be determined with the exact- 
ness required by ordinary quantitative procedure. 

FLOURS USED IN THE INVESTIGATION 

Eight flours of the higher grades (as separated in the 
process of milling) from various sources and of varying 
baking qualities were selected for the preliminary 
work. Their sources and relative baking values, as 
measured by loaf volume, are indicated in Table I. 



X d 13 

h5 2 3 



\D • • ■ • 

in lo vo lo ' 



■O O *0 \0 VO 'O vO 






Ov 00 r^ 00 1^ a> 00 



1^ 



)io r^ lo 



to bj S ^ 



s;5 






O ooo O r^ 

. r^ CN r^ fO >o vo ^ 

-H ^ (N CN CN ^ ^ 



O H 
Z O 

P9 H 



o 



4J 1^ 71 



(8^ 

.5 > I" ^^ t> 



w 

^7 T; OJ rt O 



a -9 

aot> 
xjat> 
III cs . 













ja f 






ft t£ 






SSf^ 










3 





D 





our 

ten, Lo 
b Glute 
d" Soft 



yi(UaJ4Jja>ifellJ 

d, pi< PL, fL, HI > ; fl. 



V • -- 00 On O ^^ -^ »0 CN 
rO O f^ ro -^ -^ Ti' Tf IT) 

i*z '^•^■^•^■^•^■^•^ 

t'" pq pq pq pq pq pq fq ffl 



THE PRODUCTS OF PROTEIN HYDROLYSIS FROM ENTIRE 
FLOUR 

Osborne^ and his associates have shown that there 
are five proteins present in flour, viz., gliadin, glutenin, 
albumin, globulin and proteose, the latter being of 
little significance. The first two named compose the 
gluten, already referred to, while the others are soluble 
in dilute salt solutions and are, for the most part, 
removed in the familiar process of "washing out" 
the gluten. 

Since the proteins are, for all practical considerations, 
the only nitrogen compounds in the higher grade 
flours, it was decided to submit first, in all cases, a 
sample of the entire fiour to prolonged hydrolysis with 
strong hydrochloric acid, and determine the distribu- 
tion of nitrogen in the various units. Should the re- 
sults vary in different flours, it would be necessary to 
obtain the different proteins and ascertain their com- 
position in a similar manner. If they should show the 
same chemical constitution then they must be present 
in the flour in varying amounts to account for the differ- 
ence when analyzed collectively, as is done in the 
hydrolysis of the entire flour. That the latter is true 
has, of course, been concluded by numerous investi- 
gators who have extracted flour proteins with specific 
solvents and have found their amounts to vary widely 
in different flours. The solvents most frequently used 
are: (i) alcohol — varying from 50 to 80 per cent, and 
(2) neutral salt solutions of different concentrations. 
The former was at first thought to extract only gliadin 
while the latter was considered to remove only albumin, 
globulin and proteose. Owing to the fact that solu- 
tions of varying strengths and different methods of 
extraction have been employed by different investi- 
gators, however, their results often disagree widely, 
and in many cases even fail to support the same general 
conclusions. Furthermore, it has been found that the 
solvents mentioned above are not as specific as was 
formerly supposed, and that alcohol extracts not only 
gliadin but also considerable of the "soluble proteins," 
the material so extracted depending on the strength of 
the alcohol, while salt solutions extract some gliadin 
as well as albumin and globulin, according to the con- 
centration of the solution. Other physico-chemical 
factors undoubtedly enter as well. Olson^ states that 

1 "The Vegetable Proteins" (1912). Plimmer's, Monograph, London, 
New York, etc. 

2 "Quantitative Estimation of Salt-Soluble Proteins in Wheat Flour," 
J. Ind. Eng. Chem., 6 (1914), 212. 



"the amount of gliadin extracted by i per cent sodium 
chloride solution approximately amounts to about 
29 per cent of the total proteids," and "the nitrogen 
bodies soluble in salt solution are partly or wholly 
soluble in diluted alcohols varying with the concentra- 
tion of sodium chloride used." That a study of the 
products of hydrolysis of the flour proteins both 
collectively and individually can furnish an indica- 
tion of the proportions of these proteins in the flour, 
providing there is no difTerence in the chemical con- 
stitution of -the same proteins in different flours, is 
evident from the following considerations: the per- 
centage of ammonia nitrogen yielded on the hydrolysis 
of the individual proteins of wheat flour varies as 
follows, according to Osborne, gliadin 24.5, glutenin 
18.8, leucosin (albumin of flour) 6.8, and globulin, 
7.7. Since the figures for the ammonia nitrogen 
show wider variation than do those of any other units, 
and since also the estimation of this unit is probably 
accompanied by less error than that of any of the 
others, it may be supposed that its estimation in the 
proteins taken collectively and individually will 
indicate closely the relative amounts of the various 
proteins present, providing, as mentioned before, the 
same proteins of different flours do not vary in their 
chemical constitution. 

In determining the distribution of nitrogen in the 
entire flour, lo-gram samples were hydrolyzed for 
48 hours, and the "Hausmann" units determined. 
In the case of the entire flour the presence of a large 
amount of starch occasions a voluminous precipitate 
of "humin" material which made it impractical to 
attempt a determination of all the units of the Van 
Slyke method, since a large enough sample could not 
be used to insure the estimation of the smaller units 
with sufficient accuracy that the figures would be 
of much significance. The instructions of Van Slyke 
regarding the conditions for precipitating and washing 
the bases, however, were carefully followed. 

In determining the total nitrogen in the hydrolyzed 
mixture, the presence of large amounts of "humin" 
substances resulting from the carbohydrates, and small 
amounts of fat, necessitated the slight modification of 
Van Slyke's method suggested by Gortner.^ The 
above mentioned substances make it impossible to 

> "Studies on the Chemistry of Embryonic Growth. I. Certain 
Changes in the Nitrogen Ratios of Developing Trout Eggs," J. Am. Chem. 
Soc, 36 (1913), 632-645. 



13 

obtain an aliquot until after they have been removed 
in the processes of determining the ammonia and humin 
nitrogen. Consequently, the hydrolyzed mixture is 
evaporated in vacuo to remove most of the hydro- 
chloric acid. The ammonia is distilled off as in the 
Van Slyke process (without removing the material 
from the distillation flask from which the acid was 
evaporated off), collected in standard acid, and esti- 
mated by titration; the humin filtered, washed, and 
submitted to Kjeldahl analysis for nitrogen, and total 
nitrogen determined in aliquot portions of the filtrate 
from the humin. This, added to the ammonia nitrogen 
and the humin nitrogen, gives the total nitrogen in 
the hydrolyzed sample. No correction was made 
in any of the analyses for the solubilities of the bases 
in the solutions from which they were precipitated, 
since the same conditions were observed in all cases 
and the results are strictly comparable. 

The results given in Table I were obtained from the 
analyses of the eight samples of flour by the above- 
described process. The different flours vary signifi- 
cantly with respect to the ammonia nitrogen yielded 
on hydrolysis. The basic nitrogen or nitrogen of the 
diamino acids also shows a slight variation, this being 
inversely as the variation in ammonia nitrogen. The 
variations shown in the table are much greater than 
could possibly be due to experimental error and were 
confirmed by repeated determinations. Hence, there 
can be no doubt that these variations show actual 
characteristic differences in the nitrogen distribution 
in the different samples. 

THE DISTRIBUTION OF NITROGEN IN GLIADIN, GLUTENIN, 
AND SOLUBLE PROTEINS 

Hydrolysis of the entire flour having shown character- 
istic differences in the composition of the entire protein 
material contained in them, it appeared to be necessary 
to establish as definitely as possible whether or not 
the chemical constitution of the various individual 
proteins is the same in different flours. For this 
purpose two flours which differed widely in their 
origin, total nitrogen content, and baking strength were 
selected. Flour B401 is a typical Minnesota patent 
flour, milled from northern spring wheat, of fairly 
high nitrogen content and of good baking strength, 
while B438 is a patent biscuit flour, made from a softer 
Missouri wheat, low in total nitrogen and of poor 
baking strength. Gliadin was extracted from the 



14 
gluten of the flours with alcohol and carefully purified 
by pouring the concentrated syrup from the clear 
alcoholic extract alternately into large volumes of water 
and strong alcohol and finally digesting with absolute 
alcohol and ether, according to the method of Osborne. 
Glutenin was also prepared according to Osborne's 
method which consists, briefly, of dissolving the residue 
left after the alcohol extraction of the crude gluten in a 
dilute solution of potassium hydroxide, neutralizing 
with hydrochloric acid to precipitate the glutenin, 
decanting the liquid and further extracting the pre- 
cipitate repeatedly with alcohol to remove the re- 
maining gliadin; finally digesting with absolute alco- 
hol and ether. The preparations of glutenin in this 
work were not pure, being contaminated by small 
quantities of carbohydrates, owing to lack of facilities 
for obtaining clear extracts and filtrates at the time, 
but it is believed that all other nitrogen-containing 
bodies were removed, and that the preparations served 
the purpose of the investigation, namely, to ascertain 
whether there was any appreciable difference in the 
chemical constitution of the pure proteins. Consider- 
able quantities of each of the two flours were then 
extracted with i per cent salt solution, the extracts 
were filtered as clear as possible and concentrated 
in vacuo. These extracts and weighed quantities of 
the gliadin and glutenin were then hydrolyzedfor 48 
hours with strong HCl. The gliadin and glutenin 
were analyzed according to the Van Slyke method, 
while only ammonia nitrogen was determined in the 
case of the soluble proteins. From the results shown 
in Table 11 (i, 2, and 3) it is readily seen that, after 
making allowance for the limits of experimental error 
of the method, there is no apparent difference in the 
chemical constitution of the proteins of typical strong 
and weak flours of the same market grade. 

THE DISTRIBUTION OF NITROGEN IN CRUDE GLUTEN 

More complete evidence that the gluten-forming 
proteins are of the same chemical constitution in differ- 
ent flours was obtained by analyzing thoroughly 
washed crude glutens of three flours of widely differing 
characteristics. The same two flours as in the immedi- 
ately preceding experiments were used, and in addition, 
B444, a Kansas flour of exceedingly high nitrogen and 
gluten content, but of low baking strength, as shown 
in Table I. The results, obtained from the complete 
Van Slyke process as applied to the crude glutens from 
these three flours, appear in Table II (4) and indicate 



15 

that not only are the gluten-forming proteins in flours 
of widely differing baking qualities of the same chemical 
constitution, but the ratio of gliadin to glutenin is 
probably the same, or very nearly so, in flours of the 
same market grade but very different baking strengths. 
With respect to this latter point, it may be said that 
since, as is shown above, gliadin yields 26 per cent of 
its nitrogen as ammonia nitrogen after hydrolysis, 
while glutenin yields only 16 per cent of its nitrogen 
in this fraction, the determination of ammonia nitrogen 
of the hydrolyzed glutens will certainly indicate any 
significant variation in the ratio of gliadin to glutenin 
in different flours, although the limits of experimental 
error are not narrow enough to indicate very small 
variations in this ratio. The data in Table 11 (4) 
indicate very clearly, therefore, that there is no signifi- 
cant variation in the gliadin-glutenin ratio in flours 
of such widely varying baking strength as those used 
in this investigation. 

THE SIGNIFICANCE OF THE SOLUBLE PROTEINS IN 
AFFECTING THE NITROGEN DISTRIBUTION IN FLOUR 

It is evident that the differences in the percentages 
of ammonia nitrogen and basic nitrogen yielded on the 
hydrolysis of the several entire flours, as shown in 
Table I, cannot be accounted for as being due to 
differences in chemical composition of the individual 
proteins since it has been clearly shown that these 
have the same chemical constitution. 

It was though't at first that the varying percentages 
of starch in the different flours might cause differences 
in the percentage of ammonia nitrogen, since Mann,^ 
in 1906, states "if in addition to the carbohydrate, 
ammonia or other nitrogenous substances are in solu- 
tion, then the humins combine with the ammonia and 
thereby become nitrogenous." In order to ascertain 
whether varying proportions of starch would influence 
the results obtained by the Van Slyke method as used 
in these investigations, a sample of the flour B401, 
to which had previously been added 20 per cent of 
its weight of wheat starch,- was hydrolyzed and the 
distribution of nitrogen in the products of hydrolysis 
determined. . There was no signiflcant change in the 
percentage of ammonia nitrogen when compared with 
the sample to which no starch was added, although 
there was a very noticeable increase in humin N and 
a corresponding decrease in basic N, as shown in Table 
III. 

1 "Chemistry of the Proteids." New York, 1906. 



i6 



■<*• nl 






ob^^-* 



Tf O 



trios 



Z .B-O 



[ooj4 r^sooot^a\0 



J o 



•25H«^- 



vo ^ O CM^ ^ 



H^3« 



— Mo-*ooOvr^ — 
O" looo — vo-^^o 
'f o 



, TO -M tj 



00^ o r^ ON 1^ fN r^ 

•* 4; 

looo'i'io o 



P5^ 



o ^ 



'i^r. 



^Z 

^ ft! . 

>w 



„ OB f^ Or^>ot^io vo ■* 
^mi3 »ooo-*^o ror^ 






2 b|-b:h-s.s 



Basic 


Mono-amino 


N 


acid N 


8.10 


65.77 


6.95 


65.62 



17 

Further, it has recently been shown by Gortner and 
Blish^ that other carbohydrates than starch which 
might be present in flour do not affect the percentage 
of ammonia nitrogen obtained after hydrolysis, al- 
though they may, in many instances, increase the humin 
nitrogen by forming condensation products of humin- 
like nature with tryptophane. 

The significant variation in the percentages of am- 
monia nitrogen in the different flours (Table I) must 
therefore be due to considerable variations in the 
amounts of soluble proteins present, since the gliadin- 
glutenin ratio does not differ enough to account for 

Table III — Effect of Starch on the Distribution of Nitrogen in the 
Products of Hydrolysis of Flour B401 

Per cent of the total nitrogen 

Ammonia Humin 
Material Used N N 

B401 20.81 5.32 

B401 + 2 g. Starch 21.05 6.38 

the difference in the percentages of ammonia nitrogen. 
As already shown, the "soluble proteins" (albumin 
and globulin) yield respectively 6.8 per cent and 
7.7 per cent of ammonia nitrogen on hydrolysis, 
while the gluten yields about 23 per cent. It may be 
calculated, therefore, that flours containing the larger 
amounts of "soluble proteins" will yield the smaller 
percentages of their total nitrogen as ammonia nitrogen 
after hydrolysis. Accordingly, the flours showing 
the lower ammonia nitrogen figures as shown in Table 
I might be supposed to contain the larger percentages 
of their proteins in the form of albumin and globulin. 
Since there is no known method of quantitatively 
estimating the albumin and globulin in flour, owing 
to the fact that the extraction of the various proteins 
varies with the concentration of the solvents em^ 
ployed, the proportions of solvent to material extracted, 
and possibly other physico-chemical factors, the de- 
termination of ammonia nitrogen in hydrolyzed flour, 
flour extracts, and gluten should form a basis for a 
more exact knowledge of the proportions in which 
the various proteins occur in flours. Such methods 
are obviously unadapted to ordinary analytical use, 
but afford the best possible method of exact study 
and careful investigational work. For example, in 
Table II (3), the per cent of ammonia nitrogen yielded 
on hydrolysis of the extract with i per cent salt solu- 
tion indicates clearly that protein other than albumin 
and globulin was extracted, since, as already pointed 

1 "On the Origin of the Humin Formed by the Acid Hydrolysis of 
Proteins," J. Am. Chem. Soc, 37 (1915). 



out, the per cent of ammonia nitrogen yielded by pure 
albumin and globulin should be lower. It is suggested 
also that to ascertain how much albumin and globulin 
are extracted by alcohol of any given percentage it 
should suffice to hydrolyze the alcoholic extract and 
determine the percentage of ammonia .nitrogen in a 
similar manner. 

Since this article has been in press, the results of a 
study of the purity of proteins extracted from flour by 
various solvents, using methods here indicated, have 
been 'published by Bailey and Blish.^ 

In order to substantiate the evidence that there is 
a relation between the ammonia nitrogen yielded on 
hydrolysis of flour, and the total quantity of soluble 
proteins in the flours in question, the latter were ex- 
tracted with tap water (since it was thought desirable 
to use the same solvent as was used in washing out the 
glutens) and the percentage of "soluble nitrogen" 
in the total nitrogen of the flour estimated in the ex- 
tract. Although protein material other than globulin 
and albumin is extracted in this process, the results 
should be comparative, and in the order as indicated 
above, that is to say, the previously indicated relation- 
ship between ammonia nitrogen of the hydrolyzed 
entire flour and the soluble nitrogen should be apparent. 
That this is true is indicated by the results obtained, 
as shown in Table IV. 

Table IV — Comparison of Percentages of Soluble Nitrogen in 

Entire Flour with Ammonia Nitrogen after Hydrolysis of 

Entire Flour 

Total Per cent of total N 

Sample No. Nitrogen Soluble N Ammonia N 

B444 2.55 15.68 23.00 

B440 2.17 18.20 21.47 

B439 1.928 18.67 21.01 

B401 2.085 18.94 20.81 

B452 1.917 19.55 19.87 

B441 2.130 20.42 21.03 

B438 1.67 26.86 18.85 

B445 1.26 28,96 18.21 

Inspection of Table IV shows that, with the single 
exception of B441, as the percentage of ammonia 
nitrogen increases, the percentage of soluble nitrogen 
decreases, as was expected from the theoretical con- 
siderations already discussed. 

CONCLUSIONS 

I — The individual proteins of strong and weak 
flours are identical in their chemical constitution, 
as determined by Van Slyke's method for the analysis 
of proteins. 

[Since this went to press, the attention of the writer 

1 "Concerning the Identity of the Proteins Extracted from Wheat Flour 
by the Usual Solvents," J. Biol. Chem., 23 (1915), 345-357. 



19 

was called to a study of some of the physical constants 
of gliadin from flours of varying strengths, by Gr6h 
and Friedl,' in which they conclude that proteins of 
different flours have the same constitution.] 

II — The ratio of gliadin to glutenin is much more 
nearly constant in flours of different baking qualities 
than has heretofore been supposed. 

Ill — There is a far greater variation in the per- 
centages of the so-called "soluble proteins" (albumin 
and globulin) in flours. 

IV — Since the various proteins in the same flour 
differ widely in their content of ammonia nitrogen, 
the determination of ammonia nitrogen in flours, in 
extracts of flours made with various solvents, and in 
the crude gluten of flours, after their previous complete 
hydrolysis with strong mineral acid, can be made to 
serve as an accurate indication of the amounts of the 
various proteins present, since the proteins of widely 
different flours have been shown to have the same 
chemical constitution. 

Acknowledgment and sincere thanks are herewith 
extended to Professor R. W. Thatcher under whose 
supervision this work was done, also to Dr. R. A. 
Gortner and Professor C. H. Bailey for many valuable 
and helpful suggestions. 

Department of Agriculture 

University of Minnesota 

St. Paul, Minnesota 

> Biochem. Ztschr., 66 (1914), 154. 



BIOGRAPHICAL 

Morris J. Blish was born April 21, 1889, at Lincoln, 
Nebraska. He attended the public schools there, 
through the seventh grade. He then moved to Omaha, 
Nebraska, where he graduated from the Omaha High 
School in 1906. After working in one of the Omaha 
banks for a year, he entered the University of Nebraska 
in the fall of 1907, specializing in chemistry and grad- 
uated with the B.Sc. degree in February, 191 2. Enroll- 
ing in the graduate school of the University of Nebraska, 
he received the A.M. degree in agricultural chemistry, 
June, 1913, thesis work having been on soil chemistry 
under the direction of Dr. F. J. Alway. He received 
an appointment as research assistant in agricultural 
chemistry at the University of Minnesota, and enrolled 
in the graduate college of that university in Septem- 
ber, 1913, working under the direction of Prof. R. W. 
Thatcher. He received the Ph.D. degree in June, 
191 5, thesis work having been on the chemical consti- 
tution of the proteins of wheat flour, in relation to 
"baking strength." 



PUBLICATIONS 

1. On the Distribution and Composition of the 
Humus of the Loess Soils of the Transition Region. 

2. On the Origin of the Humin Formed by the 
Acid Hydrolysis of Proteins. (In collaboration with 
Dr. R. A. Gortner.) 

3. Concerning the Identity of the Proteins Ex- 
tracted from Wheat Flour by the Usual Solvents. (In 
collaboration with C. H. Bailey.) 



LIBRARY OF CONGRESS 




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